The goal of this proposal is to define the mechanisms by which Foxc1 and Foxc2 regulate arterial specification and lymphatic vessel development. VEGF-A signaling activates the Notch-Delta like 4 (Dll4) pathway and induces expression of Neuropilin 1 (Nrp1), a co-receptor for VEGF-A, to promote the arterial program. In contrast, the COUP-TFII nuclear receptor suppresses the arterial cell fate by inhibiting the expression of Nrp1 and Notch signaling genes. After arteriovenous diversification, a subpopulation of the venous cells acquires a lymphatic cell fate by progressively expressing Sox18 and Prox1 and differentiates into lymphatic endothelial cells (LECs). Prox1/VEGF-R3+ LECs subsequently bud from the veins via paracrine VEGF-C signaling, leading to the formation of the lymphatic vasculature. We have recently demonstrated that Foxc1 and Foxc2 are essential for arterial specification by acting upstream of Notch signaling. Foxc proteins directly induce Notch signaling genes through the VEGF-A pathway. Moreover, compound Foxc1+/-;Foxc2-/- mutants exhibit a reduction in the number of Prox1+ LECs sprouting from the cardinal vein, and both Foxc genes are expressed in LECs and the surrounding mesenchyme. Our central hypothesis of this project is that Foxc1 and Foxc2 are essential for VEGF-mediated arterial cell determination and early lymphatic development. This hypothesis will be tested by: (1) determining molecular mechanisms by which Foxc1 and Foxc2 interact with the VEGF-A signaling pathway in arterial gene expression;(2) elucidating whether Foxc1 and Foxc2 regulate arterial cell identity in VEGF-R2+ endothelial progenitors;and (3) defining cell- autonomous and non-cell autonomous roles for Foxc1 and Foxc2 in lymphatic specification and the formation of the lymphatic vasculature. The mechanistic basis for a link between signaling pathways and transcriptional regulation in arterial, venous and lymphatic endothelial cells is still largely unknown. Completion of the proposed studies will define the fundamental mechanisms governing the formation of the vascular network during development. PUBLIC HEALTH RELEVANCE: Inherited disorders of the cardiovascular system are quite common in humans, but their causes and underlying developmental mechanisms are poorly understood. It is clear that mutant mice provide useful models to elucidate the molecular and cellular mechanisms of congenital cardiovascular anomalies, including arteriovenous malformations and abnormal lymphatic vessels. The proposed studies will significantly contribute to a better understanding of the causes of congenital defects associated with abnormal blood and lymphatic vessels in infants and children and gain insight into the cellular and molecular basis of human abnormalities.